Neuropsychopharmacology (2003) 28, 850–856 & 2003 Nature Publishing Group All rights reserved 0893-133X/03 $25.00 www.neuropsychopharmacology.org 5-HT1A/1B Receptor-Mediated Effects of the Selective Serotonin Reuptake Inhibitor, Citalopram, on Sleep: Studies in 5-HT1A and 5-HT1B Knockout Mice 1 1 ´ 2 1 ¨ ,1 Christelle Monaca , Benjamin Boutrel , Rene Hen , Michel Hamon and Joelle Adrien* 1INSERM U288, NeuroPsychoPharmacologie Mole´culaire Cellulaire et Fonctionnelle, CHU Pitie´-Salpeˆtrie`re-91, Boulevard de l’Hoˆpital, 75013 Paris, France; 2Center for Neurobiology and Behavior, Columbia University, New York, NY, USA Selective serotonin reuptake inhibitors (SSRIs) are extensively used for the treatment of depression. Aside from their antidepressant properties, they provoke a deficit in paradoxical sleep (PS) that is most probably mediated by the transporter blockade-induced increase in serotonin concentration in the extracellular space. Such an effect can be accounted for by the action of serotonin at various types of serotonergic receptors involved in PS regulation, among which the 5-HT1A and 5-HT1B types are the best candidates. According to this hypothesis, we examined the effects of citalopram, the most selective SSRI available to date, on sleep in the mouse after inactivation of 5- HT or 5-HT receptors, either by homologous recombination of their encoding genes, or pharmacological blockade with selective 1A 1B antagonists. For this purpose, sleep parameters of knockout mice that do not express these receptors and their wild-type counterparts were monitored during 8 h after injection of citalopram alone or in association with 5-HT1A or 5-HT1B receptor antagonists. Citalopram induced mainly a dose-dependent inhibition of PS during 2–6 h after injection, which was observed in wild-type and 5-HT1BÀ/À mice, but not in 5-HT1AÀ/À mutants. This PS inhibition was fully antagonized by pretreatment with the 5-HT1A antagonist WAY 100635, but only partially with the 5-HT antagonist GR 127935. These data indicate that the action of the SSRI citalopram on sleep in the mouse is 1B essentially mediated by 5-HT receptors. Such a mechanism of action provides further support to the clinical strategy of antidepressant 1A augmentation by 5-HT1A antagonists, because the latter would also counteract the direct sleep-inhibitory side-effects of SSRIs. Neuropsychopharmacology (2003) 28, 850–856, advance online publication, 5 March 2003; doi:10.1038/sj.npp.1300109 Keywords: sleep–wakefulness; knockout mice; 5-HT1A receptors; 5-HT1B receptors; selective serotonin reuptake inhibitor; citalopram INTRODUCTION 1995, 1999; Sharp et al, 1997; Trillat et al, 1998; Adell et al, ´ Selective serotonin reuptake inhibitors (SSRIs) are the most 2001; Malagie et al, 2001). Another well-known action of SSRIs is their effects on frequently prescribed drugs in anxiodepressive disorders. sleep and wakefulness. Indeed, in several species and Since SSRIs produce a tonic elevation of serotonin (5- hydroxytryptamine, 5-HT) levels in the extracellular space notably the rat (Pastel and Fernstrom, 1987; Ursin et al, 1989; Lelkes et al, 1994; Maudhuit et al, 1994; Neckelmann (Fuller, 1994; Malagie´ et al, 1995), their therapeutic effect is et al, 1996b), the hamster (Gao et al, 1992), and also in probably mediated, at least in part, by action at various humans (Van Bemmel et al, 1993; Hendrickse et al, 1994), levels of the serotonergic system, notably at serotonergic the most consistent action of SSRIs is a reduction of receptors (Barnes and Sharp, 1999). In particular, it has paradoxical sleep (PS), which is sometimes associated with been proposed that the antidepressant effects of SSRIs an enhancement of wakefulness (W) and a secondary would be related to desensitization of somatodendritic 5- increase in slow wave sleep (SWS) (Maudhuit et al, 1994; HT and terminal 5-HT autoreceptors (Blier et al, 1987; 1A 1B Ursin, 2002). In the same manner, systemic treatment with Artigas et al, 1994, 2001; Pineyro and Blier, 1999), which is selective agonists at 5-HT and 5-HT receptors induces induced by and participates indirectly in the increase in 5- 1A 1B HT concentration in the extracellular space (Gartside et al, an inhibition of PS and an enhancement of wakefulness, notably in rodents (Dzoljic et al, 1992; Tissier et al, 1993; Bjorvatn and Ursin, 1994; Monti et al, 1995; Bjorvatn et al, *Correspondence: Dr J Adrien, Institut National de la Sante´ et de la 1997; Boutrel et al, 1999, 2002). In contrast, inactivation of Recherche Me´dicale, INSERM U288, CHU Pitie´-Salpeˆtrie`re, 75634 5-HT and 5-HT receptors (notably in mice with genetic Paris Cedex 13, France, Tel: +33 1 40 77 97 13, Fax: +33 1 40 77 97 1A 1B 90, E-mail: [email protected] deletions targeted at these receptors) facilitates the expres- Received 18 June 2002; revised 29 August 2002; accepted 30 October sion of PS (Boutrel et al, 1999, 2002). Altogether, these data 2002 strongly suggest that 5-HT1A and/or 5-HT1B receptors, Online publication: 6 November 2002 at http://www/acnp.org/ because they are activated by extracellular 5-HT, might citations/Nppl10602426 mediate the action of SSRIs on sleep and wakefulness. 5-HT1A/1B mediated paradoxical sleep inhibition by SSRI C Monaca et al 851 However, this hypothesis has still to be validated because (i) combined treatments, the antagonist or saline was injected 5-HT1A receptor antagonists were reported to be unable to 15 min prior to citalopram. For baseline data, mice were prevent the effects of SSRIs on sleep or wakefulness injected with saline only, in single or combined treatment, (Bjorvatn et al, 1992; Neckelmann et al, 1996a) and (ii) to as appropriate. A washout period of at least 3 days was date, no study has been published with respect to the allowed between two consecutive treatments. One series of possible involvement of 5-HT1B receptors in PS inhibition animals was used to analyze the effects of citalopram alone, by SSRIs. while another one was used in experiments with combined These considerations led us to address this question by treatments. In both series, each animal received all doses using knockout mice that do not express 5-HT1A or 5-HT1B and compounds, in a counter-balanced fashion. However, receptors (Saudou et al, 1994; Ramboz et al, 1998). We some recordings were discarded from the results because of analyzed the effects of citalopram, the most selective SSRI insufficient quality. Thus, the number of tests finally currently available (Hyttel, 1982; Milne and Goa, 1991), on contributing to the data was comprised between 4 and 10. sleep–wakefulness cycles in 5-HT1A and 5-HT1B knockout mice and their wild-type counterparts. In addition, we Polygraphic Recording investigated whether pharmacological blockade of either 5- HT1A or 5-HT1B receptors with selective antagonists could Polygraphic sleep monitoring was started just after the prevent the effects of citalopram on sleep in the mouse. injection(s) and continued during 8 h thereafter. MATERIALS AND METHODS Data Analysis and Statistics Recordings were scored manually every 15 s epoch using the Animals classical criteria for mice (Boutrel et al, 2002). The effects of All the procedures involving animals and their care were each treatment on the states of vigilance (W, SWS, PS) were conducted in conformity with the institutional guidelines analyzed for every 2 h period after injection, and were that are in compliance with national and international laws expressed (mean 7 SEM) as minutes and as percentage of and policies (council directive 87–848, October 19, 1987, baseline (injection of saline). Statistical analyses were Ministe`re de l’Agriculture et de la Foreˆt, Service ve´te´rinaire performed using ANOVA for the factors treatment and de la sante´ et de la protection animale, permissions 75–116 strain of mice. In case of significance, this ANOVA was to MH and 75–125 to JA). All mice were of the same 129/Sv followed by a post hoc Fisher test or a paired Student’s t-test genetic background. They were produced in the laboratory as appropriate, in order to determine statistical significance from homozygous breeding of wild-type and mutant (5- of the effect of each dose of a given compound. HT1AÀ/À and 5-HT1BÀ/À) strains (Saudou et al, 1994; Ramboz et al, 1998; Boutrel et al, 1999, 2002), and housed in Chemicals standard animal care facilities (see below). WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperaziny- l]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide) Surgery (0.5 mg/kg i.p.) was obtained from Wyeth Research At 2–3 months of age (body weight: 21–27 g), male wild-type (Princeton, NJ, USA); GR 127935 (20-methyl-40-(5-methyl- and mutant 5-HT1AÀ/À and 5-HT1BÀ/À mice were [1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid[4-meth- implanted under sodium pentobarbital anesthesia (70– oxy-3-(4-methyl-piperazine-1-yl)-phenyl]amide) (1 mg/kg 75 mg/kg i.p.) with the classical set of electrodes for i.p.) was from Glaxo-Wellcome (Ware, UK); citalopram polygraphic sleep monitoring, as previously described (1–10 mg/kg i.p.) was from Lundbeck (Copenhagen, DK). (Boutrel et al, 1999). In brief, EEG electrodes were inserted through the skull onto the dura over the right cerebral cortex (2 mm lateral and 4 mm posterior to bregma) and RESULTS over the cerebellum (at midline, 2 mm posterior to lambda), Pharmacological Blockade of the Serotonin Transporter EOG electrodes were positioned subcutaneously on each side of the orbit, and EMG electrodes were inserted into the In wild-type mice, blockade of the 5-HT transporter by neck muscles. All electrodes were anchored to the skull with citalopram induced a significant decrease in PS amounts, super-bond cement (Limoge-Lendais et al, 1994), and which lasted for 2–6 h after the injection (Figure 1).